Granulocyte colony stimulating factor (G-CSF) is a hematopoietic growth factor that stimulates the proliferation and differentiation of hematopoietic precursor cells and the activation of mature neutrophils. G-CSF is capable of supporting neutrophil proliferation in vitro and in vivo. The human form of G-CSF was cloned by groups from Japan and the USA in 1986 (see e.g. Nagata et al. (1986) Nature 319: 415-418). The natural human glycoprotein exists in two forms, one having 174 and the other having 177 amino acids. The more abundant and more active 174 amino acid form has been used in the development of pharmaceutical products by recombinant DNA technology.
Large quantities of recombinant G-CSF have been produced in genetically engineered Escherichia coli and have been successfully used in clinical applications to treat cancer patients suffering from chemotherapy-induced neutropenia. Escherichia coli-produced
G-CSF is a 175 amino acid polypeptide chain containing an extra methionine at its N-terminus. This protein has been produced by expressing a G-CSF gene in E. coli and purifying the protein product to homogeneity. It is a hydrophobic protein that has five cysteine residues, four of them are involved in disulfide bonding. The free cysteine residue is generally implicated in the formation of higher molecular weight aggregates upon storage in solution. Aggregates of the proteins can also be formed from oxidized forms of the protein that arise by oxidation of the internal methionine residues in the primary sequence of the protein. Of the four methionine residues, one is at the N-terminus and the other three are internal. The oxidized forms of the protein containing oxidized methionine at position 122 can be separated from the native protein and the forms containing oxidized methionine at positions 127 or 138 by reverse phase HPLC separation procedures (the positions are calculated for the methionyl-G-CSF consisting of 175 amino acids).
Filgrastim is a recombinant human G-CSF synthesized in an E. coli expression system (international non-proprietary name, INN). The structure of filgrastim differs slightly from that of the natural glycoprotein. Lenograstim (INN) is another form of recombinant human G-CSF and is synthesized in Chinese hamster ovary (CHO) cells. Filgrastim and lenograstim are marketed in Europe under the trade names Neupogen® and Granocyte, respectively. The commercially available forms of recombinant human G-CSF have a short-lived pharmacological effect and often must be administered more than once a day for the duration of the leukopenic state.
Protein-engineered variants of human G-CSF are known, e.g. those described in WO 01/87925, EP 0 456 200 A, U.S. Pat. No. 6,166,183, U.S. Pat. No. 6,004,548, U.S. Pat. No. 5,580,755, U.S. Pat. No. 5,582,823, U.S. Pat. No. 5,675,941, U.S. Pat. No. 5,416,195, U.S. Pat. No. 5,399,345, WO 2005/055946 and WO 2006/074467.
Modification of human G-CSF and other polypeptides so as to introduce at least one carbohydrate chain in addition to those in the native polypeptide has also been reported (U.S. Pat. No. 5,218,092).
In general, the stability of proteins can be improved and the immune response against these proteins reduced when these proteins are coupled to polymeric molecules. WO 94/28024 discloses that physiologically active proteins modified with PEG exhibit reduced immunogenicity and antigenicity and circulate in the bloodstream considerably longer than unconjugated proteins, i.e. have a reduced clearance rate.
The attachment of synthetic polymers to the peptide backbone to improve the pharmacokinetic properties of glycoprotein therapeutics has been explored. An exemplary polymer conjugated to peptides is PEG. The use of PEG to derivatize peptide therapeutics can reduce the immunogenicity of the peptides. For example, U.S. Pat. No. 4,179,337 discloses non-immunogenic polypeptides such as enzymes and peptide hormones coupled to PEG or poly(propylene glycol) (PPG). In addition to reduced immunogenicity, the clearance time in circulation of PEG-modified polypeptides is prolonged due to the increased size of the PEGylated polypeptide conjugate.
In addition, polymer modifications of native human G-CSF, including attachment of poly(ethylene glycol) (PEG) groups, has been reported (see, e.g., U.S. Pat. No. 5,824,778, U.S. Pat. No. 5,824,784, WO 96/11953, WO 95/21629 and WO 94/20069). Pegfilgrastim (INN) is a covalent conjugate of recombinant methionyl human G-CSF (filgrastim) and a single 20 kDa monomethoxy-PEG-molecule. The monomethoxy-PEG-molecule is covalently bound to the N-terminal methionyl residue of filgrastim. Pegfilgrastim is marketed in Europe under the trade name NEULASTA®.
The principal mode of attachment of PEG, and its derivatives, to peptides is a non-specific bonding through a peptide amino acid residue (see e.g. U.S. Pat. No. 4,088,538, U.S. Pat. No. 4,496,689, U.S. Pat. No. 4,414,147, U.S. Pat. No. 4,055,635 and WO 87/00056). Another mode of attaching PEG to peptides is through the non-specific oxidation of glycosyl residues on a glycopeptide (see e.g. WO 94/05332).
In these art-recognized methods, PEG is added in a random, non-specific manner to reactive residues on a peptide backbone. Random addition of PEG molecules has its drawbacks, including a lack of homogeneity of the final product, and the possibility that the biological or enzymatic activity of the peptide will be reduced. Therefore, efforts have been made to develop more site specific methods for attaching a synthetic polymer or other label to a peptide and it has been found that specifically conjugated, homogeneous peptide therapeutics can be produced in vitro through the action of enzymes. These enzyme-based conjugation strategies have the advantages of regioselectivity and stereoselectivity. Two principal classes of enzymes used in the synthesis of conjugated peptides are glycosyltransferases (e.g. sialyltransferases, oligosaccharyltransferases, N-acetylglucosaminyltransferases) and glycosidases. These enzymes specifically attach substrate sugars to polypeptides, which can be subsequently modified with a polymer or other moiety. Alternatively, glycosyltransferases and modified glycosidases can be used to directly transfer modified sugars to a peptide backbone (see e.g. U.S. Pat. No. 6,399,336 and US 2003/0040037, US 2004/0132640, US 2004/0137557, US 2004/0126838 and US 2004/0142856). Methods combining both chemical and enzymatic synthetic elements are also known (see e.g. US 2004/137557).
Various methods of conjugating polypeptides like G-CSF with polymeric moieties like PEG are described in the art. The preparation of glycoPEGylated G-CSF is, for example, described in WO 2005/055946. WO 2006/074467 describes the preparation of conjugates between G-CSF and PEG moieties. In this method the conjugates are linked via an intact glycosyl linking group, which is interposed between and covalently attached to the G-CSF polypeptide and the PEG moiety. The conjugates are formed from both glycosylated and unglycosylated G-CSF polypeptides by the action of a glycosyltransferase on a PEGylated substrate nucleotide sugar. The glycosyltransferase ligates a modified sugar moiety onto either an amino acid or glycosyl residue on the polypeptide. The disclosure of WO 2005/055946 and WO 2006/074467 are explicitly incorporated herein by reference in their entirety for all purposes.
Besides PEG, other polymeric moieties are useful conjugation partners with G-CSF. For example, WO 02/09766 discloses, inter alia, biocompatible protein-polymer compounds produced by conjugation of biologically active protein with a biocompatible polymer derivative. The biocompatible polymer is a highly reactive branched polymer, and the resulting conjugates contain a long linker between the polymer and polypeptide. Examples of biocompatible polymers according to WO 02/09766 are PEG, PPG, polyoxyethylene (POE), polytrimethylene glycol, polylactic acid and its derivatives, polyacrylic acid and its derivatives, polyamino acids, polyurethane, polyphosphazene, poly(L-lysine), polyalkylene oxide (PAO), water-soluble polymers such as polysaccharide, dextran, and non-immunogenic polymers such as polyvinyl alcohol and polyacryl amide.
WO 96/11953 describes N-terminally chemically modified protein compounds and methods for their production. Specifically, G-CSF compositions are described which result from coupling a water-soluble polymer to the N-terminus of G-CSF. Examples of water-soluble polymers listed in WO 96/11953 are copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), poly(n-vinyl pyrrolidone)polyethylene glycol, PPG homopolymers, polypropylene oxide/ethylene oxide copolymers or polyoxyethylated polyols.
WO 97/30148 describes polypeptide conjugates with reduced allergenicity, comprising a polymeric carrier molecule having two or more polypeptide molecules coupled thereto. These conjugates are produced by activating a polymeric carrier molecule, reacting two or more polypeptide molecules with the activated polymeric carrier molecule and blocking of residual active groups on the conjugate. This publication lists a variety of polymeric carrier molecules, including natural or synthetic homopolymers such as polyols, polyamines, polycarboxylic acids and heteropolymers comprising at least two different attachment groups.
WO 03/074087 relates to a method of coupling proteins to a starch-derived modified polysaccharide. The binding between the protein and the polysaccharide, hydroxyalkyl starch, is a covalent linkage which is formed between the terminal aldehyde group or a functional group resulting from chemical modification of the terminal aldehyde group of the starch molecule and a functional group of the protein. Disclosed protein reactive groups include amino groups, thio groups and carboxy groups.
WO 2005/014050 describes the preparation of conjugates of hydroxyalkyl starch (HAS) and a G-CSF protein, wherein at least one functional group of the reacts with at least one functional group of the protein, thereby forming a covalent linkage. Other documents disclosing HASylation, e.g., HESylation, of polypeptides include WO 2005/014655, WO 2005/092390, WO 2007/031266, WO 2005/092928 and WO 2005/092391.
Although approaches for modifying therapeutic polypeptides such as G-CSF with polymeric moieties to prolong polypeptide clearance time and to reduce immunogenicity, scant literature is available regarding developing advantageous formulations for such polymer-G-CSF-conjugates.
The above mentioned NEULASTA® (pegfilgrastim) product is a liquid composition intended for subcutaneous injection. The preparation comprises pegfilgrastim, sodium acetate, sorbitol, polysorbate 20 and water for injection and has a pH of 4.0 (see www.neulasta.com, and ROTE LISTE 2007). The NEULASTA® (pegfilgrastim) and NEUPOGEN® (filgrastim) products, both marketed by Amgen, are almost identical with respect to buffer agent, excipients and pH value of the solution: NEUPOGEN® comprises filgrastim (instead of pegfilgrastim), sodium acetate, sorbitol, polysorbate 80 and water for injection with a pH of 4.0 (see www.neupogen.com, and ROTE LISTE 2007).
Although some pharmaceutical compositions developed for non-conjugated G-CSF are presented in the patent literature in such a way as to encompass preparations in which the non-conjugated G-CSF is replaced by a PEG-G-CSF conjugate, it is obvious that the compositions are tailored to, and tested for, unconjugated G-CSF only. These references do not disclose the formulation of a glycoPEGylated G-CSF conjugate.
For example, WO 2005/042024 describes stable pharmaceutical compositions comprising G-CSF having a pH value above 4.0 and further comprising an acid. The composition is free from surfactants. The pharmaceutical composition described in WO 2005/042024 was developed for non-conjugated G-CSF; however, mention is made of its use with G-CSF chemically modified with a polymer, showing the same or improved biological activity.
Another example is WO 2005/039620 which is directed to a stable aqueous G-CSF containing composition. The composition contains succinic acid or tartaric acid or salts thereof as buffer agents and has a preferred pH in the range of 4.0 and 5.8. According to the specification, the G-CSF protein may also be synthetically modified, e.g. by enzymatic glycosylation or chemical PEGylation.
EP 1 260 230 A1 discloses stable protein formulations containing tryptophan as a stabilizer. The list of proteins covers G-CSF, and G-CSF chemically modified with PEG or the like as well. The G-CSF formulations are mentioned as preferably having a pH of 5-7, more preferably 6.0-6.7.
Another example is EP 1 336 410 A1, which describes injectable pharmaceutical formulations containing a physiologically active protein as an active ingredient and at least one sugar as a soothing agent and a pH of 6.5-7.4.
EP 1 329 224 A1 describes a G-CSF solution formulation containing at least one amino acid or a salt thereof, preferably methionine, as a stabilizer. The G-CSF solution formulations preferably have a pH of 5-7, more preferably 5.5-6.8. G-CSF chemically modified with PEG or the like is said to be also included.
The formulations described in the patent literature have only been developed and tested for unconjugated G-CSF. Though exemplary references disclosing G-CSF formulations mention the use of the formulation with a polymeric conjugate of G-CSF or a generic PEG-G-CSF conjugate, none of the references describe a formulation of a polymeric G-CSF having a particular structure.
The problem underlying the present invention is to provide a polymer-G-CSF conjugate formulation which is particularly adapted to such conjugates and which is stable at elevated temperatures, generally, above refrigerator temperature (e.g., between about 2 and about 8° C.). Further, it is an object of the invention to provide a pharmaceutical composition which does require reconstitution at any stage of its preparation and which causes as little irritation as possible when administered to a patient.